P987: A DIFFERENT BALANCE IN OXIDATIVE STRESS RESPONSE IN CALR AND JAK2 MUTATED MYELOFIBROSIS PATIENTS CORRELATES WITH CLINICAL OUTCOME
Authors: E. Genovese, M. Mirabile, S. Rontauroli, S. Sartini, S. Fantini, L. Tavernari, M. Maccaferri, P. Guglielmelli, E. Bianchi, S. Parenti, C. Carretta, S. Mallia, S. Castellano, C. Colasante, M. Balliu, N. Bartalucci, R. Palmieri, T. Ottone, B. Mora, L. Potenza, F. Passamonti, M. T. Voso, M. Luppi, A. M. Vannucchi, E. Tagliafico, R. Manfredini
Journal: HemaSphere (2022)

Background: Myelofibrosis (MF) is the Philadelphia-negative myeloproliferative neoplasm characterized by the worst prognosis and poor response to conventional therapy. Driver mutations in JAK2 and CALR impact on JAK-STAT pathway activation but also on the production of reactive oxygen species (ROS). ROS play a pivotal role in inflammation-induced oxidative damage to cellular components including DNA, that leads to greater genomic instability and promotes cell transformation. Aims: In order to unveil the role of driver mutations in oxidative stress response, we evaluated oxidative stress markers levels in CD34+ hematopoietic stem/progenitor cells and plasma from MF patients. Methods: Firstly, we have measured the levels of ROS, 8-hydroxy-2-deoxy-guanosine (8-OHdG) and activity of superoxide dismutase (SOD) in CD34+ cells from 20 JAK2 and 14 CALR myelofibrosis (MF) patients compared with 17 healthy donors (HD). In particular, the redox-sensitive CM-H2DCFDA was used to measure the intracellular ROS. The levels of 8-OHdG were detected by enzyme-linked immunosorbent assay (ELISA). Moreover, SOD activity was measured by means of colorimetric assay. Subsequently, we evaluated whether a different oxidative status could be detected in 129 plasma samples of MF patients (n=86 JAK2, n=43 CALR), as the level of total antioxidant capacity (TAC), an analyte frequently used to assess the antioxidant status of biological samples. Finally, we evaluated the association between TAC plasma levels and overall survival (OS) by means of multivariate analysis. Results: Our results demonstrated that ROS production in CD34+ cells from CALR-mutated MF patients is strongly increased compared with patients harboring JAK2 mutation, leading to increased oxidative DNA damage. Moreover, CALR-mutated cells show less SOD antioxidant activity than JAK2-mutated ones. Subsequently, we found that high TAC plasma levels correlate with detrimental clinical features, such as high levels of lactate dehydrogenase (LDH) and circulating CD34+ cells. Moreover, high TAC plasma levels are also associated with a poor OS in JAK2-mutated patients. Multivariate analysis demonstrated that high plasmatic TAC is an independent prognostic factor for OS that allows the identification of patients with inferior OS in both DIPSS lowest and highest categories. Summary/Conclusion: Altogether, our results confirmed that CALR mutation has a higher impact on the oxidative stress status in MF cells compared to JAK2 variant. Furthermore, plasma samples from CALR mutated patients have significantly lower TAC levels leading to a lower responsiveness to oxidative injury. On the other hand, increased TAC levels correlate with the presence of JAK2 mutation and several detrimental clinical features. MF patients with high plasmatic TAC display inferior survival and multivariate analysis demonstrated that increased TAC activity might represent a novel prognostic biomarker independent from DIPSS classification. We speculated that the high increase in oxidative stress in CALRmutated patients could be involved in the activation of protective mechanisms which ultimately promote cell death. On the contrary, in JAK2 mutated patients, the slight increase in oxidative stress can determine the persistence of cells with damaged DNA where the accumulation of mutations promotes the disease progression.